JP2000074880A - Sample introducing method for microchip - Google Patents

Abstract

PROBLEM TO BE SOLVED: To restrict troubles caused by delay and dispersion in switching of voltage impressing directions. SOLUTION: In this sample introducing method, one end of a sample flow passage 4 is made to have 1.3 kV of potential, the other end is grounded (GND), one end of an analytical passage 5 is made to have 1.1 kV of potential, the other end is made to have 1.9 kV of potential, and a sample is moved to the vicinity of a crossing part 6 (A). Then, a potential of the passages 4, 5 are reduced to one-tenth to reduce electroendsmosis rate and a moving speed of the sample, and the sample is guided to the crossing part 6 (B). A voltage impressing direction is switched to make one end of the passage 5 have 0.11 of potential, to bring the other end into GND, and to make both ends of passage 4 floating potentials (C). Since the electroendsmosis rate and the moving speed of the sample are small, spreading of a sample plug and excessive moving are restricted even when delay and dispersion in switching of the voltage impressing directions are generated.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for introducing a sample of a microchip in an electrophoresis apparatus for analyzing a very small amount of sample with high speed and high separation. A microchip in which a sample flow path intersecting an analysis flow path is formed, and a hole reaching the analysis flow path or the sample flow path is formed at a position corresponding to the analysis flow path and the sample flow path on one surface of the transparent plate member. In the sample introduction method.

[0002]

2. Description of the Related Art In the case of analyzing a very small amount of protein, nucleic acid, or the like, an electrophoresis method has been conventionally used, and a capillary electrophoresis apparatus is an example of a technique for realizing the apparatus. A capillary electrophoresis device is a device in which a glass capillary having an inner diameter of 100 μm or less is filled with an electrophoresis buffer, a sample is introduced at one end side, and a high voltage is applied between both ends to develop an analyte in the capillary. is there.
Since the inside of the capillary has a large surface area relative to the volume, that is, high cooling efficiency, a high voltage can be applied, and a very small amount of sample such as DNA can be analyzed at high speed and with high resolution.

In recent years, DJ Harrison et al./Science 261 (19) has been proposed as a form that can be expected to increase the speed of analysis and reduce the size of the apparatus in place of a glass capillary that is complicated to handle.
93) 895-897 and Anal. Chim. Acta 283 (1993) 361-366.
As shown in (2), a chip (microchip) used for capillary electrophoresis formed by joining two substrates has been proposed. Figure 1 shows an example of the microchip.
Shown in A pair of transparent substrates (generally, glass, quartz, resin, etc.) 1 and 2 are formed, and a sample flow path 4 and an analysis flow path 5 are formed on the surface of one substrate 2 so as to intersect each other. The reservoir 3 is provided as a through hole at positions corresponding to both ends of the flow path 4 and the analysis flow path 5.

When using this microchip, both substrates 1 and 2 are overlapped as shown in FIG. 1C, and an electrophoresis buffer is injected into the sample channel 4 and the analysis channel 5 from one of the reservoirs 3. Then, after injecting a sample into the reservoir 3 at one end of the sample flow path 4, an electrode is inserted into each of the reservoirs 3 or both ends of the sample flow path 4 are formed using electrodes formed in the respective reservoirs 3 in advance. A predetermined high voltage is applied for a predetermined time to guide the sample to the intersection 6 between the sample channel 4 and the analysis channel 5. Next, a predetermined voltage for electrophoresis is applied to both ends of the analysis channel 5, and the sample present at the intersection 6 is guided into the analysis channel 5 and separated. By arranging a detector at an appropriate position in the analysis channel 5, the separation component is detected. Also, at intersection 6
The sample at the intersection 6 to prevent the sample from spreading
In addition, a weak voltage is applied to the analysis channel 5 when the sample is guided to the sample flow channel, and to the sample channel 4 when the sample is separated.

[0005]

Generally, it is difficult to switch the high voltage, and a delay occurs when a relay or the like is used. Often, the delay is on the order of 1 to 10 milliseconds. On the other hand, for example, when a tris-borate buffer (pH 8.2) is used, when a potential of several hundreds to several thousands V / cm is applied, the speed at which the sample is carried by the electroosmotic flow through the quartz flow path (hereinafter, referred to as “the velocity”) The electroosmotic speed) is on the order of several μm / msec. Therefore, the sample area (also referred to as a sample plug) moves several μm to several tens μm due to a delay in switching the high voltage of the relay. Generally, the size of a sample plug determined by the configuration of an electrophoresis chip is several tens of μm.
In addition, undesired phenomena such as the spread of the sample plug and a decrease in the analysis accuracy are induced due to the movement of the sample plug due to the delay in switching of the high voltage.

Accordingly, an object of the present invention is to suppress problems caused by delay or variation in voltage switching during sample injection.

[0007]

According to the method for introducing a sample of a microchip according to the present invention, an analysis flow path and a sample flow path intersecting the analysis flow path are formed inside a transparent plate-like member. Using a microchip having a hole that reaches the analysis flow path or the sample flow path at a position corresponding to the analysis flow path and the sample flow path on one surface, applying a voltage for moving the sample to both ends of the sample flow path The sample injected at one end of the sample flow path is guided to the intersection of the analysis flow path and the sample flow path, and then the voltage is switched to apply a voltage for sample separation analysis to both ends of the analysis flow path. This is a microchip sample introduction method for introducing a sample into an analysis channel, in which electroosmotic flow in the sample channel is suppressed at least at the time of switching the voltage application direction.

In the present invention, since the electroosmotic flow is suppressed when the voltage application direction is switched, the electroosmotic speed and the moving speed of the sample are reduced. As a result, even if there is a delay of about 10 milliseconds in the switching of the applied voltage direction, the sample hardly moves in practice and does not affect the analysis accuracy.

[0009]

DESCRIPTION OF THE PREFERRED EMBODIMENTS One mode of a method for suppressing electroosmotic flow when switching a voltage application direction is to apply a voltage for moving a sample to both ends of a sample flow path and to inject the voltage to one end of the sample flow path. When a sample is migrated toward the intersection of the analysis channel and the sample channel, when the sample approaches the intersection, the voltage is reduced to guide the sample to the intersection. Theoretically, the rate of electroosmosis is proportional to the voltage. In this aspect, before switching the voltage application direction, the voltage for moving the sample is reduced, so that the electroosmotic speed and the moving speed of the sample when switching the voltage application direction can be reduced.

Another embodiment of the method of suppressing the electroosmotic flow when switching the voltage application direction is a method of adding a thickener to at least one of the sample solution and the migration buffer.
In theory, since the electroosmotic speed is inversely proportional to the viscosity, the electroosmotic speed and the moving speed of the sample can be reduced.

[0011] Still another embodiment of the method for suppressing the electroosmotic flow when switching the voltage application direction is to cool the sample channel. By cooling the sample solution and the migration buffer, the viscosity of the sample solution and the migration buffer increases, so that the electroosmotic speed and the moving speed of the sample can be reduced.

Still another embodiment of the method for suppressing the electroosmotic flow at the time of switching the voltage application direction is to use a sample solution and a migration buffer having a low pH. Electroosmotic flow mobility with pH in fused silica capillaries was determined by KD Lukacs et al. In J. High Resolut. Chromat.
ogr. Chromatogr. Commun., 8, 407 (1985), it is known that at pH 3 the value is about an order of magnitude smaller than at pH 8. When using a microchip whose surface condition changes with the pH of the solution, such as a microchip made of fused silica, the pH of the sample solution
Can be adjusted to adjust the electroosmotic speed and the sample moving speed.

[0013]

FIG. 2 is a schematic diagram showing the operation of one embodiment. In this example, IEEE MEMS '97 Proceedings, 299
A quartz electrophoresis chip prepared according to (1997) was used. The configuration of the chip is the same as that shown in FIG. This embodiment will be described with reference to FIG. (A) The sample channel 4, the analysis channel 5, and the reservoir 3 are filled with an electrophoresis buffer, the sample is injected into the reservoir 3 at one end of the sample channel 4, and then the sample channel 4 on the side into which the sample is injected is filled. A voltage of 1.3 kV is applied to one end, and the other end is set to GND potential. In addition, 1.
A voltage of 1 kV and a voltage of 1.9 kV are applied to the other end, and the
(In the analysis channel 5, the base end in the electrophoresis direction is one end, and the end in the traveling direction is the other end.) The sample moves in the sample flow path 4 and is guided to the vicinity of the intersection 6 after a predetermined voltage application time has elapsed.

(B) The voltages applied to both ends of the sample channel 4 and the analysis channel 5 are reduced to 1/10, respectively. As a result, since the electroosmotic speed and the moving speed of the sample are theoretically proportional to the voltage, the electroosmotic speed and the moving speed of the sample are reduced. In this embodiment, the voltages applied to both ends of the sample flow path 4 and the analysis flow path 5 are respectively 1/10, but the voltage reduction ratio in the present invention is not limited to 1/10.

(C) After the sample reaches the intersection 6,
The voltage application direction is switched by a relay (not shown), and the voltage at one end of the analysis flow path 5 is kept at 0.11 kV, and the voltage at the other end is set to the GND potential. At this time, the sample flow path 4
To a floating potential. Since the electroosmotic speed and the moving speed of the sample are low, even if the switching or delay in the switching of the voltage application direction occurs, the spread and extra movement of the sample plug can be suppressed.

(D) With the other end of the analysis channel 5 kept at the GND potential, the voltage at one end is increased to 1.0 kV. At this time,
A voltage of 0.13 kV is applied to both ends of the sample flow path 4 to suppress the sample from diffusing into the analysis flow path 5. (E) Next, the voltage applied to the sample flow path 4 is increased to 0.6 k.
V to moderately suppress the diffusion of the sample into the analysis channel 5. After the sample led to the intersection 6 is separated in the analysis channel 5, it is detected by a detector at a detection position at the other end of the analysis channel 5. In this embodiment, the applied voltage at the time of switching the voltage application direction is reduced to adjust and decrease the electroosmotic speed and the moving speed of the sample. Problems such as extra movement can be suppressed, and a decrease in analysis accuracy can be suppressed.

FIG. 3 is a diagram showing a state when a sample is introduced in this embodiment. (A) A voltage for moving the sample is applied to both ends of the sample flow path 4, a sample plug of the sample introduced from the a side of the sample flow path 4 is formed at the intersection 6, and the voltage for moving the sample is set to 1 for each. / 10 (FIG. 2 (A),
(B)). (B) When both ends of the sample flow path 4 were set to the floating potential and a voltage for separation and analysis was applied to the analysis flow path 5 and the voltage application direction was switched, the sample plug at the intersection 6 remained stationary. (FIG. 2 (C)).

(C) When a voltage for preventing sample plug diffusion is applied to both ends of the sample channel 4 and a voltage for separation and analysis is continuously applied, the sample at the intersection 6 maintains the original sample plug shape. And started moving to the d side (Fig. 2
(D)). (D), (E) When the voltage for preventing diffusion of the sample plug was increased and the voltage for separation and analysis was continuously applied, a clean sample plug was introduced to the d side of the separation channel 5 (FIG. 2).
(E)).

FIG. 4 is a diagram showing a state at the time of sample introduction in the conventional sample introduction method. (A) A voltage for moving the sample was applied to both ends of the sample flow path 4, and a sample plug of the sample introduced from the a side of the sample flow path 4 was formed at the intersection 6. (B) When the voltage for separation analysis is applied and the voltage application direction is switched, the ON / OFF variation of the relay causes
The sample at the intersection 6 slightly moved to the c side and spread. In the figure, the sample moves to the c side, but sometimes moves to the d side, and the moving direction is not necessarily determined. (C) to (D) Thereafter, when the voltage for separation analysis was continuously applied, the sample plug spread in the analysis flow path direction was introduced to the d side of the separation flow path 5.

As a method of controlling the electroosmotic speed and the moving speed of the sample, a viscosity may be increased by adding a thickener such as hydroxypropylcellulose to the sample solution.
In theory, the rate of electroosmosis and the rate of sample movement are inversely proportional to viscosity. Increasing the viscosity of the sample solution can decrease the electroosmotic speed and the moving speed of the sample. As the thickener, for example, 5% of a 2% solution from Sigma
Hydroxypropylcellulose reagents that sell at 0 centipoise, 100 centipoise, or 4000 centipoise are sold. The viscosity of a commonly used electrophoresis buffer is about 1 centipoise at 20 ° C., which is almost the same as that of water.

FIGS. 5A and 5B are schematic views showing a microchip used for another method of adjusting the electroosmotic speed and the moving speed of the sample, wherein FIG. 5A is a perspective view, and FIG. 5B is a perspective view of AA ′ in FIG. It is sectional drawing along a position. The same parts as those in FIG. 1 are denoted by the same reference numerals, and description thereof will be omitted. A groove 7 formed by, for example, dicing is formed at a position corresponding to the sample flow path 4 on a surface of the substrate 2 opposite to the surface on which the sample flow path 4 and the analysis flow path 5 are formed. An aluminum block 8 machined so as to fit into the groove 7 is inserted into 7. Oil is applied between the groove 7 and the aluminum block 8 to improve heat conduction. The surface of the aluminum block 8 opposite to the groove 7 is joined to a Peltier cooler 9.

The sample channel 4 is cooled by the Peltier cooler 9 via the aluminum block 8 to such an extent that the sample solution and the electrophoresis buffer filled in the sample channel 4 are not frozen. As a result, the viscosities of the sample solution and the migration buffer are increased, the electroosmotic speed and the moving speed of the sample when switching the voltage application direction can be reduced, and problems such as the spread and extra movement of the sample plug can be suppressed. Such an effect by cooling is particularly effective not only when a normal electrophoresis buffer is used but also when a thickener is added as described above.

When using a chip whose surface condition changes depending on the pH of the solution, such as a fused silica microchip, the pH of the sample solution and the electrophoresis buffer is lowered to reduce the electroosmotic speed and the moving speed of the sample. It may be adjusted. By lowering the pH of the sample solution and the electrophoresis buffer as described above, the electroosmotic speed and the moving speed of the sample at the time of switching the voltage application direction can be reduced, and problems such as spreading of the sample plug and extra moving are suppressed. be able to.

The embodiments of the present invention have been described above. However, the present invention is not limited to the above embodiments, and various changes may be made within the scope of the present invention described in the appended claims. be able to. Examples of embodiments of the present invention will be illustrated below.

(1) An analysis flow path and a sample flow path intersecting the analysis flow path are formed inside the transparent plate member, and correspond to the analysis flow path and the sample flow path on one surface of the transparent plate member. Using a microchip having a hole reaching the analysis channel or the sample channel at a position, applying a voltage for moving the sample to both ends of the sample channel, and injecting the sample into one end of the sample channel. To the intersection of the analysis flow path and the sample flow path, and thereafter, switching the voltage application direction, applying a voltage for sample separation analysis to both ends of the analysis flow path, and introducing the sample into the analysis flow path. In the microchip sample introduction method, when the sample approaches the intersection, the voltage for moving the sample is reduced to guide the sample to the intersection, and at least when the voltage application direction is switched. The method for introducing a sample into a microchip according to claim 1, wherein an electroosmotic flow in the sample channel is suppressed.

(2) An analysis flow path and a sample flow path intersecting the analysis flow path are formed inside the transparent plate member, and correspond to the analysis flow path and the sample flow path on one surface of the transparent plate member. Using a microchip having a hole reaching the analysis channel or the sample channel at a position, applying a voltage for moving the sample to both ends of the sample channel, and injecting the sample into one end of the sample channel. To the intersection of the analysis flow path and the sample flow path, and thereafter, switching the voltage application direction, applying a voltage for sample separation analysis to both ends of the analysis flow path, and introducing the sample into the analysis flow path. In a method for introducing a sample into a microchip, a thickener is added to at least one of a sample solution and an electrophoresis buffer, and at least at the time of switching of the voltage application direction, electrolysis is performed in the sample channel. A method for introducing a sample into a microchip, characterized in that permeation is suppressed.

(3) An analysis flow path and a sample flow path intersecting the analysis flow path are formed inside the transparent plate member, and correspond to the analysis flow path and the sample flow path on one surface of the transparent plate member. Using a microchip having a hole reaching the analysis channel or the sample channel at a position, applying a voltage for moving the sample to both ends of the sample channel, and injecting the sample into one end of the sample channel. To the intersection of the analysis flow path and the sample flow path, and thereafter, switching the voltage application direction, applying a voltage for sample separation analysis to both ends of the analysis flow path, and introducing the sample into the analysis flow path. The method for introducing a sample of a microchip according to claim 1, wherein the sample flow path is cooled, and the electroosmotic flow in the sample flow path is suppressed when the voltage application direction is switched. Sample introduction method.

(4) An analysis flow path and a sample flow path intersecting the analysis flow path are formed inside the transparent plate member, and correspond to the analysis flow path and the sample flow path on one surface of the transparent plate member. Using a microchip having a hole reaching the analysis channel or the sample channel at a position, applying a voltage for moving the sample to both ends of the sample channel, and injecting the sample into one end of the sample channel. To the intersection of the analysis flow path and the sample flow path, and thereafter, switching the voltage application direction, applying a voltage for sample separation analysis to both ends of the analysis flow path, and introducing the sample into the analysis flow path. In the microchip sample introduction method, a low pH sample solution and a migration buffer are used, and the electroosmotic flow in the sample flow path is suppressed when the voltage application direction is switched. Microchip sample introduction method.

[0029]

In the microchip sample introduction method according to the present invention, the electroosmotic flow in the sample flow path is suppressed at least at the time of switching the voltage application direction. In addition, the moving speed of the sample is reduced, and problems such as the spread or extra movement of the sample plug due to the delay or variation in switching of the voltage application direction can be suppressed, and a decrease in analysis accuracy can be suppressed.

[Brief description of the drawings]

FIGS. 1A and 1B are diagrams showing a microchip, wherein FIGS. 1A and 1B are plan views showing a transparent plate member constituting the microchip, and FIG. 1C is a front view of the microchip.

FIG. 2 is a schematic diagram illustrating an operation of one embodiment.

FIG. 3 is a diagram showing a state at the time of sample introduction in the embodiment.

FIG. 4 is a diagram showing a state at the time of sample introduction in a conventional sample introduction method.

5A and 5B are schematic views showing a microchip used in a method for adjusting the electroosmotic speed and the moving speed of a sample according to another embodiment, wherein FIG. 5A is a perspective view and FIG. 5B is an AA ′ line of FIG. It is sectional drawing along a position.

[Explanation of symbols]

 1, 2 glass substrate 3 reservoir 4 sample flow path 5 analysis flow path 6 intersection

Claims (1)

[Claims] An analysis flow path and a sample flow path intersecting the analysis flow path are formed inside a transparent plate member, and a position corresponding to the analysis flow path and the sample flow path on one surface of the transparent plate member. Using a microchip formed with a hole reaching the analysis channel or the sample channel, applying a voltage for moving the sample to both ends of the sample channel, the sample injected into one end of the sample channel The sample is guided to the intersection of the analysis flow channel and the sample flow channel, and then the voltage is applied to the sample flow channel by switching the voltage application direction and applying a voltage for sample separation analysis to both ends of the analysis flow channel. A method for introducing a sample into a microchip, wherein an electroosmotic flow in the sample flow path is suppressed at least at the time of switching the voltage application direction.